HOT MCT Techniques.

Elo Elo eh eh eh. Haha.

Hey guys wassap. Its been 7 cool/hot weeks since sip started. Its great working in the department of experimental surgery. Its cool looking at the animals but i am doing only invitro work, therefore i do not handle the animals.

In this post i will cover alot on mammalian cell technology stuff as this is the bulk of my experiments. Hope this will also freshen your memory.

Now lets got to the basics which is subculturing of cells. As charmaine mention earlier subculture is done when the cells have reached confluence. In my lab i will subculture the cancer lines (eg. Hep G2) when it is 80-90% confluent. Let my give 2 major importance of subculturing.

1) To prevent overcrowding of cells. Therefore porvides space for the cells to grow as over crowding will result in scenescence of the cells.

2)Subculturing replenish the nutrients contained in the media. Metabolic wastes which could be harmful to the cells at high concentrations are removed during subculturation.

If the cells have not reached confluence, we have to change their media about once or twice a week depending on the cell type. Certain cell lines are slow proliferators while others grows very fast. These slow proliferators will eventually use up all the nutrients and produce waste therefore a media change is needed for them.

Here are the steps to perform subculture. It is different from what we learnt during MCT lessons.

A) Subculturing of Cells (perform in Biosafety cabinet)
1. Discard spent media from culture flask into waste bottle
2. Wash with 10mL PBS and discard
3. Add 2mL of trypsin and incubate for 2- 3 minutes
4. Observe under the inverted microscope if cells are detached
5. After cells are detached, add 8mL of media to deactivate the trypsin and mix well
6. Transfer 10mL of cell suspension into a 50mL centrifuge tube
7. Centrifuge the cell suspension at 1500 rpm for 3 minutes
8. Discard supernatant and resuspend cell pallet while washing with 10mL PBS
9. Centrifuge at 1500 rpm for 3 minutes
10. Discard supernatant
11. For a cell concentration ratio of 1:20, add 10mL of media and resuspend the pallet
12. Prepare a new culture flask adding 19.5mL media
13. Aliquot 0.5mL of cells into new 75cm2 culture flask containing media
14. Check under the inverted microscope for cells
15. Incubate cells in the 370C, 5% CO2 incubator

The difference is in Step no. 6 onwards.

In my lab, after deactivating the trypsinized cells, there is one additional stet which is the washing with PBS (step no. 8). This is done so as to remove as much trypsin as possible. That is why in our MCT lab sessions, some of us had a hard time detaching the cells with trypsin as previously, the trypsin is not fully removed by an additional washing step. The cells are adapting to the enzyme trypsin effect and thus it takes a longer time for the trypsin to work for subsequent trypsinization. Also in my lab, culture flask are changed every time subculturing is done. I guess our school do not do such as it is too costly and it is not for something of high importance such as research. Changing flask regularly helps prevents contamination as older flasks are exposed to the environment which contains many microbes.

Now let me talk about our favourite hemocytometer. Guess what it is used for? Anyone?

Correct! It is used for cell counting. For mammalian cells, cell counting is done for many reasons. Examples are when freezing cells, performing MTT assay and many more. For me i had to perform cell counting as I need to do MTT assay.

For cell counting, I obtain the cell number estimate by counting big(1mm by 1mm) squares at the 4 corners of the counting chamber.

*Sorry ppl im unable to put a nice/hot photoshoot of the counting chamber cos blogger is being a pain for now.*

Trypan blue is used in cell counting to differentiate the dead cells from the living ones. The dead cells will be stained blue whereas viable cells will look white under the microcope. Formula for cell concentration:

C=n x 10^4 x df

Whereby,

c=cell concentration (cells/ml)

n=average no. of cells/mm^2 (average of the 4 squares)

10^4=volume counted

df=dilution factor

Cell counting is crucial in MTT assay as seeding of cells in each of the wells of the 96-well plate must contain equal amounts of cells so that accurate results will be produced when the assay is performed.

Ok guys my post ends here and pls do ask any questions if you are in doubt. Have fun and hope you guys learn alot during SIP. Take care.

Najib Bin Hamid (0503217B)
TG01